The excessive production and accumulation of beta-amyloid (Abeta) peptides within the brain is believed to initiate the pathological cascade culminating in Alzheimer Disease (AD). Autosomal dominant forms of AD appear to cause disease by promoting Abeta production, especially Abeta42. Abeta is derived from the proteolytic cleavage of beta-amyloid precursor protein (betaAPP) by beta- and gamma-secretase activities. Inhibiting either secretase is a major goal in AD therapeutics. Beta-secretase was recently identified, gamma-secretase, which is responsible for Abeta42 generation, has not been identified, though presenilin 1 was shown to be a central component of gamma-secretase which probably consists of a complex of proteins. Thus, the further elucidation of a gamma-secretase complex, as well as the discovery of proteins (genes) that influence either beta- or gamma-secretase activities, are major goals in AD research. Random Homozygous Knockout (RHKO) is a genetic approach designed to discover genes based on their biological function and has been successfully used to identify genes involved in tumorigenesis, ubiquitin-mediated protein degradation and resistance to infectious agents. Specifically, RHKO accomplishes random inactivation of both alleles of chromosomal genes by using gene search (retroviral) vector cassettes that contain a regulated antisense promoter. Hence, an inactivated gene can later be turned on to validate the knockout. Also, a tag is included in the vector so the gene can be easily isolated. A recent version of RHKO causes transfected cells to produce circularized plasmids containing the targeted gene, which can be used to transform bacteria for rapid DNA cloning. A modified version of RHKO will be used to identify genes whose inactivation alters beta- and gamma-secretase activities in several newly developed assay systems. The effects of such genes on Abeta generation will be validated in a tetracycline regulated cell culture systems. Cell biological characteristics of the gene products will be studied in cell culture systems and in transgenic mouse models. The results are expected to illuminate genetic pathways of Abeta generation and its regulation, and to allow evaluation of the diagnostic and therapeutic potential of the newly identified genes. Specific Aim 1 proposes to identify gamma-secretase gene or genes affecting gamma- or beta-secretase activities. Specific Aim 2 is to validate and characterize the genes identified and to understand how these gene products may affect betaAPP metabolism.